(414a) Surface-Capped Protein Nanoparticles for Non-Viral Gene Delivery

Gene therapy holds great promise for addressing critical genetic diseases and cancers. The modification or correction of disease-causing genetic mutations through the delivery of nucleic acids is of high interest. Successful delivery of nucleic acids such as plasmids (pDNA) and messenger RNA, to the cells requires overcoming certain biological barriers; these include nuclease degradation, short half-life in the bloodstream, and internalization into target cells. Nanoparticles (NPs) have played a significant role in advancing the field of non-viral gene delivery. However, clinical translation remains a challenge for most NP systems. These challenges are predominantly due to poor NP stability, inefficient nucleic acid encapsulation, delivery to the desired cells, and effective transfection within the cells. Protein nanoparticles (PNPs) are a promising platform for nanotherapeutics, offering biological and physical properties suitable for overcoming barriers to clinical translation. This work explores the surface stabilization of PNPs with polycations to improve NP stability and enhance pDNA transfection both in vitro and in vivo.

Utilizing electrohydrodynamic (EHD) jetting, serum albumin was used to encapsulate a large pDNA. The as-jetted PNPs were collected in an aqueous polycation solution, resulting in the layer-by-layer deposition of positively charged nanoparticles, with the polycation stabilizing the negatively charged protein nanoparticle surface. These nanoparticles are called surface-capped PNPs (scPNPs). Electron microscopy and dynamic light scattering (DLS) confirmed that the scPNPs have sub-200 nm diameters in their dry and hydrodynamic states. Moreover, the scPNP size and positive charge were stable in aqueous conditions for more than 2 weeks. A quantification method utilizing nanoparticle tracking analysis (NTA) and UV-visible absorbance, was established to determine the pDNA content per scPNP, which indicated an overall yield of 75-80%. Cellular uptake kinetics of scPNPs were evaluated in two different cell lines, and 100% cellular uptake was achieved within 6 hours of incubation. In human HepG2 cells, transfection rates of 50% were attained by varying the pDNA amounts per scPNP and scPNP dosing, with negligible cytotoxicity observed. Preliminary results in a mouse glioblastoma model indicate that scPNPs demonstrate higher transfection efficacy than JetPEI®, a commercial transfection agent.

This work aims to advance the fundamental understanding of design choices when optimizing the efficiency of large nucleic acid internalization, delivery, and transfection efficacy. The scPNP platform promises to incorporate a variety of payloads – such as siRNA, mRNA, antisense oligonucleotides, and Cas9-sgRNA complexes – for an expansion into numerous therapeutic applications tackling diverse cancers and genetic disorders.